Gene therapy remains a promising treatment for AIDS because of the limited effectiveness of existing antiviral agents such as immunotherapy, protease inhibitors and vaccines. Several types of RNA gene therapies have been developed and shown to inhibit HIV-1 replication in mammalian cell cultures; these include antisense RNA, catalytic RNA (ribozymes) and high-affinity RNA ligands (apamers or decoys). However, their efficiencies appear to be highly variable and, in many cases, ineffective.
RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants initiated by double-stranded RNA that is homologous in sequence to the silenced gene. (Sharp, P. A., 2001) This powerful genetic technology has recently been shown to be useful in mammalian cells (Elbashir, S. M., et al., 2001; Novina, C. D., et al., 2002; Brummelkamp, T. R., et al., 2002; Paddison, P. I., et al., 2002; Lee, N. S., et al., 2002). One form of double-stranded RNA, known as small interfering RNA (siRNA), has been shown to target and inhibit expression of HIV-1 genes. (Lee, N. S., et al., 2002)
It is known that dsRNA≧30 bp can trigger interferon responses in mammalian cells that are intrinsically sequence-nonspecific to the inducing dsRNA. (Elabashir, S. M., et al., 2001) Duplexes of 21-nucleotides (siRNAs) with overhanging 3′ ends can mediate RNAi in a sequence-specific manner in cultured mammalian cells. (Elbashir, S. M., et al., 2001)
In mammalian systems, the sequence-specific RNAi effect can be observed by introducing siRNAs into target cells directly by transfection (Elbashir, S. M., et al., 2001 and Novina, C. D., et al., 2002), or indirectly by endogenous expression of siRNAs or short hairpin siRNAs (shRNAs). (Brummelkamp, T. R., et al., 2002; Paddison, P. I., et al., 2002; and Lee, N. S., et al., 2002). The stem-loop parts of the shRNAs parallel the naturally occurring stem-loop structures that are processed by the Dicer to yield small temporal RNAs (stRNAs) or microRNAs (miRNAs). Therefore, expression of shRNAs or miRNA precursors corresponding to HIV-1 sequences could be acted upon by the human Dicer to yield HIV-specific siRNAs. Recently, expression of several shRNAs having different stem-loop structures from DNA templates in mammalian cells silenced several targets as effectively as synthetic siRNAs. (Brummelkamp, T. R., et al., 2002; and Paddison, P. I., et al., 2002) However, it remains unclear what makes the best RNA silencing hairpin.
Inhibition of HIV-1 replication using synthetic siRNAs or siRNAs expressed from plasmid DNAs has shown that siRNA technology may be useful as a therapeutic strategy to inhibit HIV-1 replication and infection in host cells. (Novina, C. D., et al., 2002; Paddison, P. I., et al., 2002; and Jacque, J.-M., et al. 2002) These data provide a basis for investigating therapeutic uses of siRNAS as anti-HIV-1 agents. For this purpose, siRNAs are expressed in cells to confer immunity to HIV-1 infection and prevent viral replication.
One approach for expressing siRNAs in cells involves inserting siRNA genes into a viral vector to be transduced into primary cells. Vector-based strategies that target combinations of viral genes and/or cellular genes should provide an improvement to RNA-based antiviral therapeutics. CCR5, an HIV-1 co-receptor, provides an attractive target candidate because homozygous deletions in CCR5 effectively confer protection from HIV-1 without any serious deleterious effects in immune functions in humans. A vector-based strategy for silencing HIV-1 rev in human cells has been demonstrated using the U6 promoter system. (Paddison, P. I., et al., 2002).
The basic design of the VA1 vector is described in Cagnon and Rossi, Antisense and Nucleic Acid Drug Development 10:251-261, 2000, incorporated herein by reference. The use of the adenoviral VA1 promoter for ribozyme expression is described in U.S. Pat. No. 6,100,087 (Rossi et al.), which also is incorporated herein by reference.
Vector-based siRNA-directed gene silencing in primary cells is lower than that seen in cell lines possibly because of poor expression of siRNAs or lower efficiency of silencing machinery in primary cells.
Strategies are needed to improve the expression and efficacy of silencing RNAs in primary cells. Strategies include alternative promoter systems for expressing interfering RNA molecules, and particularly siRNAs, in primary cells. The present invention addresses this need.